CN105758838B - The detection of induced with laser enhancing Raman spectrum and method for separating and its device in liquid - Google Patents
The detection of induced with laser enhancing Raman spectrum and method for separating and its device in liquid Download PDFInfo
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- CN105758838B CN105758838B CN201610220457.2A CN201610220457A CN105758838B CN 105758838 B CN105758838 B CN 105758838B CN 201610220457 A CN201610220457 A CN 201610220457A CN 105758838 B CN105758838 B CN 105758838B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N2021/653—Coherent methods [CARS]
- G01N2021/655—Stimulated Raman
Abstract
The invention discloses detection and method for separating and its devices that induced with laser in a kind of liquid enhances Raman spectrum.The present invention forms three-dimensional ligh trap, and enriched in metals nano particle is overlapped by exciting light with induction optical focus, forming induced with laser enhances Raman signal in the surface of sample to be tested in a liquid by inducing light to focus on the sample to be tested of suspension；The implementation method of the present invention is easy, and spectrum enhancing effect is apparent, and the biological sample in-situ Raman spectrum being suitable in liquid environment quickly detects；Sample to be tested is needed to be incubated jointly with metal nanoparticle, especially suitable for field of fast detection such as disease, epidemic situation, food securities；Toxic damages of the metal nanoparticle to biological sample are greatly reduced, so as to farthest react living cells actual biological state；The optical tweezer that the present invention is formed can be sorted, extracts and be eluted, and realized Raman spectrum identification and the sorting function of single living cell, be conducive to the follow-up study of biological sample.
Description
Technical field
The present invention relates to Raman signal sample detection technologies, and in particular to induced with laser enhances Raman spectrum in a kind of liquid
Detection and method for separating and its device.
Background technology
When light beam is irradiated on sample, scattering light overwhelming majority photon is emitted in the form of Rayleigh scattering, is scattered
Light is equal with incident light frequency.Additionally, there are the photons of more few parts, and Raman scattering will occur, these photons and sample phase
Energy exchange is generated when interaction, the frequency of emergent light can occur blue shift or red shift, be referred to as Raman shift (Raman
Shift), the two corresponds respectively to anti-Stokes (anti-Stokes) line and Stokes (Stokes) line.In practical study
Usually Raman diffused light is collected with spectrometer form Raman spectrum.Relative to fluorescence research side the most commonly used in biological study
Method, the most prominent advantage right and wrong label of Raman spectrum, sensitivity height, abundant information very narrow without photobleaching, spectrum line, Ke Yiding
The chemical constituent and content of amount reflection sample, are known as " molecular fingerprint identification technology ".And normal Raman scattered signal is extremely micro-
It is weak, be research, application development maximum restraining factors.
For the very weak problem of Raman signal, currently, having developed the method for a series of enhancing Raman signal, specifically include
Two classes.The first kind is to be based on local surface plasma resonance (Localized Surface Plasmon Resonance, LSPR)
The surface-enhanced Raman (Surface Enhanced Raman Spectroscopy, SERS) and point enhancing Raman (Tip of effect
Enhanced Raman Spectroscopy,TERS).According to enhancing principle, sample to be tested needs and can generate plasma effect
Base material have physical distance close enough.It is realized under vacuum conditions so SERS, TERS are often combined with electron microscope
Raman signal enhances, more to the inorganic samples research such as materials science field.This method is for the life substantially all in water environment
The sample research of object field is very limited.In recent years, a series of Raman optical spectrum methods based on SERS principles have accordingly been developed
Carry out the samples such as on-spot study microorganism, makes some progress [1,2].Currently, carrying out biological sample research using SERS, most
Main way of realization be still that SERS sensitivity nano particles are placed in sample bottom in the form of substrate, sample cannot be effectively ensured
The bioactivity of product.
The enhancing of second class Raman signal depends on nonlinear optical effect, includes specifically：Resonance raman (Resonance
Raman Spectroscopy, RRS), excited Raman (Stimulated Raman Spectroscopy, SRS), be concerned with it is anti-this
Lentor Raman (Coherent anti-Stocks Raman Spectroscopy, CARS).Such enhancing is mainly by super
Fast laser system is realized, realizes that process is relative complex, is required instrument hardware, experimental implementation higher.
Currently, method most like with the present invention in being studied biological sample using SERS technologies is to use colloidal form
Gold or the samples such as silver nano-grain and biology be directly mixed into solution, then dripped to measure substrate sheet on, after to be dried
Directly measure.Common substrate has calcirm-fluoride, silicon, aluminium flake etc..The parameter that experiment changes generally comprises nano-particle colloid
Size, particle aggregation form etc..It can guarantee that sample to be tested and nano particle have physical distance close enough and increase to reach after drying
Strong effect.The feature of the method maximum is that realization process is easy, and enhancing effect is most important to consider standard.
Metallic particles (thin layer) is directly synthesized in sample surfaces or cell wall, is another SERS Enhancement Methods.It closes
Enhance LSPR effects at various types of nano particles, nanoscale ditch (gap), or particle progress three-dimensional group is filled with expansion
The contact area of sample and metal surface.In addition, will synthesize on metal nanoparticle " spreading " to sample；In nano-metal particle table
Bread is by the watchcase of good biocompatibility；Culture is incubated jointly with biological sample to realize that endocytosis etc. is all the SERS developed recently
Method.
Invention content
For the above problems of the prior art, the present invention proposes induced with laser enhancing Raman light in a kind of liquid
The detection of spectrum and method for separating and its device.
An object of the present invention is to provide detection and sorting sides that induced with laser in a kind of liquid enhances Raman spectrum
Method.
The detection method of induced with laser enhancing Raman spectrum, includes the following steps in the liquid of the present invention：
1) sample preparation：
Prepare sample to be tested, and prepares metal nanoparticle, it is after evenly mixing by sample to be tested and metal nanoparticle, molten
In the liquid being contained in transparent sample cell；
2) illumination electro-optical device is opened, illumination electro-optical device is launched bright field light, is radiated in sample cell, after object lens focus,
It respectively after the first and second dichroscopes, is imaged on imaging device, and by image transmitting to monitor, real-time display sample
Sample to be tested inside pond；
3) illumination electro-optical device is closed；
4) induction electro-optical device is opened, induction electro-optical device sends out near-infrared laser as induction light, after light combination mirror, then through the
One dichroscope enters object lens, and by the object lens high order focusing of high-NA, focus, which is located to be suspended in sample cell, waits for test sample
On product, three-dimensional ligh trap is formed in a liquid, and suspend fixed sample to be tested, and enriched in metals nano particle is in the table of sample to be tested
Face；
5) open excitation-light unit, excitation-light unit sends out the laser of visible light wave range, as exciting light, through light combination mirror with
After inducing combiner, enter object lens through the first dichroscope, is focused by the object lens of high-NA, focus is similarly positioned in sample cell
In sample to be tested on, the nano-metal particle that excitation is enriched in sample to be tested surface generates local surface plasma resonance LSPR
Effect, while sample to be tested being excited to generate Raman signal, Raman signal and local surface plasma resonance LSPR effect phase interactions
With forming induced with laser enhances Raman signal；
6) induced with laser enhancing Raman signal is focused by object lens, through the first dichroscope and induction light and excites optical branching,
Through the second dichroscope and illumination optical branching, after exciting optical filter, then line focus lens focus, into Raman spectrum
Instrument obtains induced with laser enhancing Raman spectrum；
7) Raman spectrometer sends data to computer, and analysis induced with laser enhancing Raman spectrum obtains sample to be tested
Information.The method for separating of induced with laser enhancing Raman spectrum, includes the following steps in the liquid of the present invention：
1) sample preparation：
Prepare sample to be tested, and prepares metal nanoparticle, it is after evenly mixing by sample to be tested and metal nanoparticle, molten
In the liquid being contained in transparent sample cell, sample cell is fixed on objective table；
2) illumination electro-optical device is opened, illumination electro-optical device is launched bright field light, is radiated in sample cell, after object lens focus,
It respectively through the first and second dichroscopes, is imaged on imaging device, and by image transmitting to monitor, real-time display sample cell
Internal sample to be tested；
3) illumination electro-optical device is closed；
4) induction electro-optical device is opened, induction electro-optical device sends out near-infrared laser as induction light, through light combination mirror, then through first
Dichroscope enters object lens, and by the object lens high order focusing of high-NA, focus is located at the sample to be tested being suspended in sample cell
On, three-dimensional ligh trap is formed in a liquid, and suspend fixed sample to be tested, and enriched in metals nano particle is in the surface of sample to be tested；
5) open excitation-light unit, excitation-light unit sends out the laser of visible light wave range, as exciting light, through light combination mirror with
After inducing combiner, enter object lens through the first dichroscope, by the object lens high order focusing of high-NA, focus is similarly positioned in sample
On sample to be tested in product pond, the nano-metal particle that excitation is enriched in sample to be tested surface generates local surface plasma resonance
LSPR effects, while sample to be tested being excited to generate Raman signal, Raman signal and local surface plasma resonance LSPR effect phases
Interaction, forming induced with laser enhances Raman signal；
6) induced with laser enhancing Raman signal is focused by object lens, through the first dichroscope and induction light and excites optical branching,
And through the second dichroscope and illumination optical branching, after exciting optical filter, then line focus lens focus, into Raman spectrum
Instrument obtains induced with laser enhancing Raman spectrum；
7) Raman spectrometer sends data to computer, and analysis induced with laser enhancing Raman spectrum obtains sample to be tested
Information obtains the property of sample to be tested；
8) excitation-light unit is closed；
9) illumination electro-optical device is opened, illumination electro-optical device is launched bright field light, is radiated in sample cell, after object lens focus,
Enhance Raman signal branch through the first dichroscope and induction optical branching, then through the second dichroscope and induced with laser, is imaged on
On imaging device, and by image transmitting to monitor, the sample to be tested inside real-time display sample cell；
10) focus of control induction light is located on selected sample, and the three-dimensional ligh trap that light to be induced is formed in a liquid is steady
After fixed, the movement of the mobile drive sample cell of objective table is controlled, three-dimensional ligh trap relative sample is formed by control induction light
It moves in pond so that selected sample is relatively moved with sample cell, realizes sample sorting and extraction.
Wherein, a capillary is fixed in step 10), in sample cell, internal diameter is slightly larger than the size of selected sample,
One end of capillary is put into sample cell, the other end is connected with micro-syringe, when selected sample is moved to capillary in sample
When port in pond, syringe applies negative pressure, and sample that will be selected is sucked out.Further include elution before step 10) is sorted：
Control induction light is motionless, and sample to be tested, which is induced to be formed three ligh trap constraint suspensions, to be fixed in the solution, is carried out to sample cell
Perfusion wash realizes the elution of metal nanoparticle.
It is another object of the present invention to provide detections and sorting that induced with laser in a kind of liquid enhances Raman spectrum
Device.
The present invention liquid in induced with laser enhancing Raman spectrum detection include with sorting unit：It induces electro-optical device, swash
Light-emitting device, light combination mirror, the first dichroscope, object lens, sample cell, metal nanoparticle, condenser lens, excitation optical filter,
Raman spectrometer and computer；Wherein, sample to be tested is uniformly mixed with metal nanoparticle, is dissolved in and is contained in transparent sample cell
In liquid in；Induction electro-optical device sends out near-infrared laser as induction light, enters object through light combination mirror, then through the first dichroscope
Mirror, by the object lens high order focusing of high-NA, focus, which is located at, to be suspended on the sample to be tested in sample cell, is formed in a liquid
Three-dimensional ligh trap, suspend fixed sample to be tested, and enriched in metals nano particle is in the surface of sample to be tested；Excitation-light unit is sent out can
The laser of light-exposed wave band enters object lens, by height through light combination mirror with after induction combiner as exciting light through the first dichroscope
The object lens high order focusing of numerical aperture, focus are similarly positioned on the sample to be tested in sample cell, and excitation is enriched in sample to be tested table
The nano-metal particle in face generates local surface plasma resonance LSPR effects, while sample to be tested being excited to generate Raman signal,
Raman signal interacts with local surface plasma resonance LSPR effects, and forming induced with laser enhances Raman signal；Laser lures
It leads after enhancing Raman signal focuses by object lens, through the first dichroscope and exciting light and induces optical branching, filtered by exciting light
After device, then line focus lens focus, into Raman spectrometer, induced with laser enhancing Raman spectrum is obtained；Raman spectrometer is through number
It is connected to computer according to line, analysis induced with laser enhancing Raman spectrum obtains the information of sample to be tested.
Sample cell uses non-blooming transparent material.The sample for being dissolved in liquid, sample to be tested are held in sample cell
Including biological sample and abiotic particulate matter；If sample to be tested is one kind or more in cell, albumen, microorganism and chemical molecular
Kind, liquid uses one kind in buffer solution, culture solution and aqueous solution；If sample to be tested is the solid particulate matter in air, liquid
Body is aqueous solution or organic solvent.Coverslip is set at the top of sample cell, is used for covering liquid, is connect with reducing liquid-to-air
It touches and exchanges, increase the stability of testing result, especially effectively reduce error in volatility detection.
The material of metal nanoparticle is using one kind in the coinage metals such as gold, silver, platinum and copper, laser excitation local surface
Plasma resonance LSPR effects.The incident wavelength of laser be more than metal nanoparticle 10 times of diameter, metal nanoparticle it is straight
Diameter is less than 100nm.Induction light and exciting light are possible to excitation metal nanoparticle and generate local surface plasma resonance LSPR.
Induction electro-optical device include：Near infrared laser, the first electronic shutter and coupled lens group；Wherein, near-infrared laser
Device sends out near-infrared laser, and through the first electronic shutter, after being expanded by coupled lens group, the diameter of light beam is more than the rear pupil of object lens
Diameter forms induction light.Switch of first electronic shutter as induction electro-optical device, controls the switch of near infrared laser, to
Control induces the time etc. operated using laser when the induction time of light, elution separation.
Excitation-light unit includes：TEM00 mode lasers, the second electronic shutter and expand microscope group；Wherein, solid state laser is sent out
The laser for going out visible light wave range is expanded by expanding microscope group, forms exciting light, the second electronic shutter is arranged in expanding microscope group.The
Switch of two electronic shutters as excitation-light unit, controls the switch of solid state laser, to control the Raman scattering of exciting light
Firing time.First electronic shutter and the second electronic shutter are manually controlled by remote controler.
Further, the invention also includes illumination electro-optical device and imaging device, the side opposite with object lens is set outside sample cell
Illumination electro-optical device is set, and imaging device is set with object lens homonymy outside sample cell；Illumination electro-optical device launches bright field light, irradiates
In sample cell, transmitted light is after object lens focus, and through the first dichroscope and induction optical branching, then through the second dichroscope and swashs
Photoinduction enhances Raman signal branch, is imaged on imaging device, imaging device is connected to monitor, real-time display through data line
Sample to be tested inside sample cell.Imaging device is connected to computer by data line, realizes the control to imaging device.Imaging
Device uses CCD camera.
Advantages of the present invention：
The present invention forms three-dimensional ligh trap, metal is made to receive in a liquid by inducing light to focus on the sample to be tested of suspension
Rice grain is enriched in the surface of sample to be tested, by exciting light with induction optical focus overlaps, collective effect, at the same excite LSPR with
Raman signal, Raman signal generates induced with laser with LSPR interactions enhances Raman signal enhancing effect, thus more convenient
To the information of sample to be tested；The implementation method of the present invention is easy, and spectrum enhancing effect is apparent, the biology being suitable in liquid environment
Sample in-situ Raman spectrum quickly detects；The present invention does not need sample to be tested and is incubated jointly with metal nanoparticle, can effectively carry
High conventional efficient reduces consumptive material consumption, especially suitable for field of fast detection such as disease, epidemic situation, food securities；Simultaneously as
Enhancement effect can effectively reduce the intensity and Spectral acquisition times of exciting light, reduce the influence that laser generates sample；This
Outside, metal nanoparticle and sample to be tested action time are short in method of the invention, greatly reduce metal nanoparticle to life
The toxic damages of object sample, so as to farthest react living cells actual biological state.
The non-contact suspension of sample to be tested is fixed in liquid further more, near-infrared laser is lossless, while again receiving metal
Rice grain is enriched to the surface of sample to be tested, keeps the pool wall of sample to be tested and sample cell contactless, effectively reduces bottom slide
Interference of the autofluorescence to Raman signal；Meanwhile avoiding mechanical injuries to sample to be tested.
Importantly, entire detection process is all rapidly completed in liquid environment, it is (as thin in lived to biological sample
Born of the same parents) it is almost lossless, be conducive to the researchs such as further cultivate, expand after Raman spectrum detects, to waiting for after raman spectroscopy measurement
The optical tweezer that sample can be formed easily with the present invention is sorted, extracted and is eluted, and realizes the Raman light of single living cell
Spectrum discrimination and sorting function are conducive to the follow-up study of biological sample.
Description of the drawings
Fig. 1 enhances the schematic diagram of the detection and sorting unit of Raman spectrum for induced with laser in the liquid of the present invention；
Fig. 2 is that the one embodiment for the detection method that induced with laser enhances Raman spectrum in the liquid according to the present invention obtains
Induced with laser enhance Raman spectrogram.
Specific implementation mode
Below in conjunction with the accompanying drawings, by specific embodiment, the present invention is further explained.
As shown in Figure 1, the detection of induced with laser enhancing Raman spectrum includes with sorting unit in the liquid of the present embodiment：It lures
Guiding device 1, light combination mirror 3, the first dichroscope 4, object lens 5, sample cell 6, metal nanoparticle, focuses thoroughly excitation-light unit 2
Mirror, Raman spectrometer 7, illumination electro-optical device 8 and computer 9；Wherein, held in transparent sample cell 6 be dissolved in it is to be measured in liquid
Sample, metal nanoparticle are injected into the liquid of sample cell；Induction electro-optical device 1 sends out near-infrared laser as induction light, first
Through light combination mirror 3 after the first speculum M1 reflection, object lens 5 are reflected by the first dichroscope 4, by 5 high order focusing of object lens, from
The bottom of sample cell focuses in sample cell 6, and focus, which is located at, to be suspended on the sample to be tested in sample cell 6, and three-dimensional ligh trap is formed,
Suspend fixed sample to be tested, and enriched in metals nano particle is in the surface of sample to be tested；Excitation-light unit 2 sends out visible light wave range
Laser, as exciting light, first after the second speculum M2 reflection through light combination mirror 3 with after induction combiner, by the first dichroic
Mirror 4 is reflected into object lens, and by 5 high order focusing of object lens, focus is similarly positioned on the sample to be tested in sample cell, and excitation, which is enriched in, to be waited for
The nano-metal particle on sample surface generates local surface plasma resonance LSPR effects, while sample to be tested being excited to generate drawing
Graceful signal, Raman signal interact with local surface plasma resonance LSPR effects, and forming induced with laser enhances Raman signal；
Induced with laser enhancing Raman signal is collected by object lens 5, is transmitted through the first dichroscope 4, the second dichroscope 83 reflection, through third
Speculum M3 reflections, after excitation optical filter 71 filters Rayleigh scattering light, line focus lens 72 focus, into Raman spectrometer
7, obtain induced with laser enhancing Raman spectrum；Raman spectrometer 7 is connected to computer 9 through data line, and what Raman scattering was surveyed is point
The vibration signal of sub-key, by analyzing the Raman spectrum measured, can further deducing the ingredient of sample to be tested and containing relatively
Amount.Light combination mirror 3 uses dichroscope.Optical filter 71 is excited to use notch filter.
In the present embodiment, it is punched among the organic plastics that sample cell 6 is about 5mm using thickness, quartzy wave is pasted in bottom
Piece 62, quartzy Cover Glass 61 in top cover.Sample to be tested is pyridine, and liquid uses the silver sol of 1mL concentrations, be added 10uL,
The NaCl salting liquids of 1M, final concentration are about 10mM.
For the material of metal nanoparticle using silver, the diameter about 60nm of silver nano-grain is spherical.
Induction electro-optical device 1 include：Near infrared laser 11, the first electronic shutter 12 and coupled lens group 13；Wherein, close red
Outer laser 1 sends out the single transverse mode laser of wavelength 1064nm, through the first electronic shutter 12, after being expanded by coupled lens group 13, light
The diameter of beam is more than the diameter of the rear pupil of object lens, forms induction light.
Excitation-light unit 2 includes：Solid state laser 21, the second electronic shutter 22 and expand microscope group 23；Wherein, Solid State Laser
Device 21 sends out 532nm (500mW) TEM00 mould laser, expands 23 by expanding microscope group, forms exciting light, be arranged in expanding microscope group
Second electronic shutter 22.
The setting of electro-optical device 8 is illuminated in the upper surface of sample cell, illumination electro-optical device includes bright field illumination halogen lamp 81 and condenser
82, bright field illumination halogen lamp 81 launches bright field light, is radiated in sample cell 6 after being focused by condenser 82, and sample is poly- through object lens 5
It is defocused, it transmits, is imaged on imaging device 84, imaging device 84 is through data through the first dichroscope 4 and the second dichroscope 83
Line is connected to monitor 85, the sample to be tested inside real-time display sample cell.Imaging device 84 is connected to calculating by data line
Machine 9 realizes the control to imaging device.Imaging device uses CCD camera.
The detection method of induced with laser enhancing Raman spectrum includes in the liquid of the present embodiment：
1) sample preparation：
A) prepared by metallic particles：Metal nanoparticle is present in using silver, silver nano-grain in silver sol；
B) sample preparation：The pyridine of the 99% pure concentration of analysis dilutes 10 times, prepares the NaCl solution of 1M；
C) by 10uL NaCl solutions, 8uL pyridines, which inject in 1mL silver sols, to be uniformly mixed；
D) sample cell, covered are injected.
2) illumination electro-optical device is opened, illumination electro-optical device is launched bright field light, is radiated in sample cell, after object lens focus,
Respectively after the transmission of the first and second dichroscopes, it is imaged on imaging device, and by image transmitting to monitor, real-time display
Sample to be tested inside sample cell.
3) illumination electro-optical device is closed.
4) induction electro-optical device is opened, induction electro-optical device sends out 1064nm laser as induction light, after light combination mirror, then through the
One dichroscope is reflected into object lens, and by the object lens high order focusing of high-NA, focus, which is located to be suspended in sample cell, to be waited for
In sample, 5~10 μm away from sample cell bottom, three-dimensional ligh trap is formed, enriched in metals nano particle is in the surface of sample to be tested.
5) excitation-light unit is opened, excitation-light unit sends out 532nm (500mW) TEM00 mould laser, as exciting light, warp
Light combination mirror enters object lens, by the object lens high order focusing of high-NA, focus is same with after induction combiner through the first dichroscope
Sample is located on the sample to be tested in sample cell, and the nano-metal particle that excitation is enriched in sample to be tested surface generates local surface etc.
Ion resonance LSPR effects, while sample to be tested being excited to generate Raman signal, Raman signal and local surface plasma resonance
LSPR effects interact, and forming induced with laser enhances Raman signal.
6) induced with laser enhancing Raman signal is collected by object lens, through the first dichroscope and induction light and excites optical branching,
Through the second dichroscope and illumination optical branching, after exciting optical filter, then line focus lens focus, into Raman spectrum
Instrument obtains induced with laser enhancing Raman spectrum.Raman spectrum is received by the subsidiary liquid nitrogen refrigerating spectrum CCD of spectrometer.
7) Raman spectrometer sends data to computer, and analysis induced with laser enhancing Raman spectrum obtains sample to be tested
Ingredient and relative amount.
Fig. 2 is one group of data that the induced with laser enhancing Raman spectroscopy device of the present embodiment is acquired.X indicates to draw in Fig. 2
Graceful displacement, unit cm-1；Y-axis indicates Raman scattering light intensity, arbitrary unit.Smooth solid line indicates induced with laser enhancing Raman spectrum letter
Number, the band warning triangle solid line close to X-axis indicates spontaneous Raman spectrum signal.Spontaneous Raman group replaces nano silver with distilled water
Grain, other are consistent with experimental group.
Under the induced with laser enhancing Raman spectroscopy device of the present invention, Pyridine Molecules enhance Raman to be believed with spontaneous Raman spectrum
It number compares, enhances more than two orders of magnitude.
It is finally noted that the purpose for publicizing and implementing example is to help to further understand the present invention, but this field
Technical staff be appreciated that：Without departing from the spirit and scope of the invention and the appended claims, various to replace and repair
It is all possible for changing.Therefore, the present invention should not be limited to embodiment disclosure of that, and the scope of protection of present invention is to weigh
Subject to the range that sharp claim defines.
Claims (10)
1. in a kind of liquid induced with laser enhancing Raman spectrum detection method, which is characterized in that the detection method include with
Lower step：
1) sample preparation：
Prepare sample to be tested, and prepare metal nanoparticle, after evenly mixing by sample to be tested and metal nanoparticle, is dissolved in Sheng
It is placed in the liquid in transparent sample cell；
2) illumination electro-optical device is opened, illumination electro-optical device is launched bright field light, is radiated in sample cell, after object lens focus, respectively
It after the first and second dichroscopes, is imaged on imaging device, and by image transmitting to monitor, in real-time display sample cell
The sample to be tested in portion；
3) illumination electro-optical device is closed；
4) induction electro-optical device is opened, induction electro-optical device sends out near-infrared laser as induction light, after light combination mirror, then through the one or two
Enter object lens to Look mirror, by the object lens high order focusing of high-NA, focus, which is located at, to be suspended on the sample to be tested in sample cell,
Three-dimensional ligh trap is formed in a liquid, and suspend fixed sample to be tested, and enriched in metals nano particle is in the surface of sample to be tested；
5) excitation-light unit is opened, excitation-light unit sends out the laser of visible light wave range, as exciting light, through light combination mirror and induction
After combiner, enter object lens through the first dichroscope, is focused by the object lens of high-NA, focus is similarly positioned in sample cell
On sample to be tested, the nano-metal particle that excitation is enriched in sample to be tested surface generates local surface plasma resonance LSPR effects
It answers, while sample to be tested being excited to generate Raman signal, Raman signal interacts with local surface plasma resonance LSPR effects,
Forming induced with laser enhances Raman signal；
6) induced with laser enhancing Raman signal is focused by object lens, through the first dichroscope and induction light and optical branching is excited, through the
Two dichroscopes and illumination optical branching, after exciting optical filter, then line focus lens focus, into Raman spectrometer,
Obtain induced with laser enhancing Raman spectrum；
7) Raman spectrometer sends data to computer, and analysis induced with laser enhancing Raman spectrum obtains the letter of sample to be tested
Breath.
2. the method for separating of induced with laser enhancing Raman spectrum in a kind of liquid, which is characterized in that the method for separating, including with
Lower step：
1) sample preparation：
Prepare sample to be tested, and prepare metal nanoparticle, after evenly mixing by sample to be tested and metal nanoparticle, is dissolved in Sheng
It is placed in the liquid in transparent sample cell, sample cell is fixed on objective table；
2) illumination electro-optical device is opened, illumination electro-optical device is launched bright field light, is radiated in sample cell, after object lens focus, respectively
It through the first and second dichroscopes, is imaged on imaging device, and by image transmitting to monitor, inside real-time display sample cell
Sample to be tested；
3) illumination electro-optical device is closed；
4) open induction electro-optical device, induction electro-optical device send out near-infrared laser as induction light, through light combination mirror, then through the one or two to
Look mirror enters object lens, and by the object lens high order focusing of high-NA, focus, which is located at, to be suspended on the sample to be tested in sample cell,
Three-dimensional ligh trap is formed in a liquid, and suspend fixed sample to be tested, and enriched in metals nano particle is in the surface of sample to be tested；
5) excitation-light unit is opened, excitation-light unit sends out the laser of visible light wave range, as exciting light, through light combination mirror and induction
After combiner, enter object lens through the first dichroscope, by the object lens high order focusing of high-NA, focus is similarly positioned in sample cell
In sample to be tested on, the nano-metal particle that excitation is enriched in sample to be tested surface generates local surface plasma resonance LSPR
Effect, while sample to be tested being excited to generate Raman signal, Raman signal and local surface plasma resonance LSPR effect phase interactions
With forming induced with laser enhances Raman signal；
6) induced with laser enhancing Raman signal is focused by object lens, through the first dichroscope and induction light and excitation optical branching, and is passed through
Second dichroscope and illumination optical branching, after exciting optical filter, then line focus lens focus, into Raman spectrometer,
Obtain induced with laser enhancing Raman spectrum；
7) Raman spectrometer sends data to computer, and analysis induced with laser enhancing Raman spectrum obtains the letter of sample to be tested
Breath, obtains the property of sample to be tested；
8) excitation-light unit is closed；
9) illumination electro-optical device is opened, illumination electro-optical device is launched bright field light, is radiated in sample cell, after object lens focus, through the
One dichroscope and induction optical branching, then enhance Raman signal branch through the second dichroscope and induced with laser, it is imaged on imaging
On device, and by image transmitting to monitor, the sample to be tested inside real-time display sample cell；
10) focus of control induction light is located on selected sample, after the three-dimensional ligh trap that induction light is formed in a liquid is stablized,
The movement for controlling the mobile drive sample cell of objective table is formed by three-dimensional ligh trap relative sample pond to control induction light
It is mobile so that selected sample is relatively moved with sample cell, realizes sample sorting and extraction.
3. method for separating as claimed in claim 2, which is characterized in that fix a capillary in sample cell, capillary it is interior
Diameter is more than the size of selected sample, and one end of capillary is put into sample cell, and the other end is connected with micro-syringe, when what is selected
When sample is moved to port of the capillary in sample cell, syringe applies negative pressure, and selected sample is sucked out；In step 10)
Further include elution before being sorted：Control induction light is motionless, and sample to be tested, which is induced the three-dimensional ligh trap to be formed constraint suspension, to be fixed
In the solution, perfusion wash is carried out to sample cell, realizes the elution of metal nanoparticle.
4. the detection of induced with laser enhancing Raman spectrum and sorting unit in a kind of liquid, which is characterized in that described to detect and divide
Screening device includes：Induce electro-optical device, excitation-light unit, light combination mirror, the first dichroscope, object lens, sample cell, metal nano
Grain, condenser lens, excitation optical filter, Raman spectrometer and computer；Wherein, sample to be tested is uniformly mixed with metal nanoparticle
It closes, is dissolved in the liquid being contained in transparent sample cell；The induction electro-optical device sends out near-infrared laser as induction light, warp
Light combination mirror, then enter object lens through the first dichroscope, by the object lens high order focusing of high-NA, focus, which is located at, is suspended in sample
On sample to be tested in pond, three-dimensional ligh trap is formed in a liquid, and suspend fixed sample to be tested, and enriched in metals nano particle is in waiting for
The surface of sample；The excitation-light unit sends out the laser of visible light wave range, photosynthetic through light combination mirror and induction as exciting light
Shu Hou enters object lens through the first dichroscope, and by the object lens high order focusing of high-NA, focus is similarly positioned in sample cell
On sample to be tested, the nano-metal particle that excitation is enriched in sample to be tested surface generates local surface plasma resonance LSPR effects
It answers, while sample to be tested being excited to generate Raman signal, Raman signal interacts with local surface plasma resonance LSPR effects,
Forming induced with laser enhances Raman signal；After the induced with laser enhancing Raman signal is focused by object lens, through the first dichroscope
It, into Raman spectrometer, is obtained after exciting optical filter, then line focus lens focus with exciting light and induction optical branching
Induced with laser enhances Raman spectrum；The Raman spectrometer is connected to computer through data line, and analysis induced with laser enhances Raman
Spectrum obtains the information of sample to be tested.
5. detection as claimed in claim 4 and sorting unit, which is characterized in that the sample cell uses non-blooming transparent material
Material.
6. detection as claimed in claim 4 and sorting unit, which is characterized in that if sample to be tested is cell, albumen, microorganism
With it is one or more in chemical molecular, the liquid use buffer solution, culture solution and aqueous solution in one kind；If sample to be tested
For the solid particulate matter in air, the liquid is aqueous solution or organic solvent.
7. detection as claimed in claim 4 and sorting unit, which is characterized in that the material of the metal nanoparticle uses
One kind in gold, silver, platinum and copper；The incident wavelength of the laser is more than 10 times of the diameter of metal nanoparticle, and the metal is received
The diameter of rice grain is less than 100nm.
8. detection as claimed in claim 4 and sorting unit, which is characterized in that the induction electro-optical device includes：Near-infrared swashs
Light device, the first electronic shutter and coupled lens group；Wherein, the near infrared laser sends out near-infrared laser, through the first electronics
Shutter, after being expanded by coupled lens group, the diameter of light beam is more than the diameter of the rear pupil of object lens, forms induction light.
9. detection as claimed in claim 4 and sorting unit, which is characterized in that the excitation-light unit includes：Solid State Laser
Device, the second electronic shutter and expand microscope group；Wherein, the solid state laser sends out the laser of visible light wave range, by expanding microscope group
It expands, forms exciting light, the second electronic shutter is set in expanding microscope group.
10. detection as claimed in claim 4 and sorting unit, which is characterized in that further include illumination electro-optical device, the second dichroic
Mirror, imaging device and monitor, opposite with the object lens side setting illumination electro-optical device outside sample cell, and outside sample cell with
Imaging device is arranged in object lens homonymy；The illumination electro-optical device launches bright field light, is radiated in sample cell, after object lens focus,
Enhance Raman signal branch through the first dichroscope and induction optical branching, then through the second dichroscope and induced with laser, is imaged on
On imaging device, the imaging device is connected to monitor through data line.
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